After almost a century of research there are still critical questions regarding the role of vitamin D in health and disease. Beyond the well established function of vitamin D in bone health, evidence is emerging that it may be a risk factor in chronic diseases ranging from autoimmune diseases and cancer to cardiovascular disease and type II diabetes. One complication in ongoing research is the lack of reliable measurement technology with sufficient selectivity and sensitivity to determine physiological levels of vitamin D and its metabolites in tissues, blood, and food. Commercially available kit assays provide high throughput analysis of 25(OH)D, but not of vitamin D, and inter-laboratory performance is poor. Additionally, these kits are unable to accurately and separately measure 25(OH)D3 and 25(OH)D2, as they are confounded by cross-reactivity with catabolic 24,25(OH)2D metabolites. On the other hand, HPLC coupled with mass spectrometry (LC-MS) offers both increased sensitivity and selectivity and minimizes interferences commonly seen from complex food matrices. Several LC-MS/MS methods have been developed for measuring vitamin D metabolites in plasma that reported Limits of Quantification (LOQ) ranging from 0.17 to 6.5 ng/mL. While promising, the premature fragmentation of vitamin D molecules in ion sources contributes to the higher variability of these methods and high LOQ. To overcome this problem, a specific vitamin D derivatization reaction based on Diels-Alder cycloaddition was developed. Using PTAD derivatization and methylamine, LOQs of 10 - 20 pg/mL have been accomplished for five vitamin D metabolites. Although a significant improvement, this method does not achieve the sensitivity of immuno- assays and does not allow for simultaneous analysis of multiple samples in a single chromatographic run. A solution to these problems that will be developed in this proposal is to increase both the selectivity and sensitivity with which the components of vitamin D are determined. This will be achieved with a new liquid chromatography-mass spectrometry approach in which the 9,10- secosteroid components of vitamin D are derivatized with a new reagent that both differentially codes components isotopically according to sample origin and greatly increases their ionization efficiency. Taken together this will enable multiple samples to be analyzed simultaneously at the level comparable to immuno-assays (1-5 pg/mL) while still allowing molecular discrimination between vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3. PUBLIC HEALTH RELEVANCE: A new quantification strategy and reagents for the analysis of vitamin D and its different forms is being developed that will facilitate broader understanding of the various bio- regulatory functions unique to this hormone-like vitamin.